This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-term goal is to understand the molecular mechanisms regulating growth plate development. The last step in maturation of the growth plate is the differentiation of proliferative chondrocytes into hypertrophic chondrocytes, which subsequently undergoes endochondral ossification. Runx 2/Cbfa1 is a transcription factor necessary for chondrocyte differentiation and hypertrophy. HDAC4 functions as a negative regulator of chondrocyte hypertrophy by binding and inhibiting Runx 2/Cbfa1 expression in the nucleus. Our recent findings indicate that HDAC4 nuclear-cytoplasm shuttling and degradation occurs in chondrocytes, allowing chondrocyte differentiation and further hypertrophy. However, the mechanisms underlying HDAC4 shuttling and degradation are unclear. The overall hypothesis includes two parts: Hypothesis 1: HDAC4 nuclear-cytoplasmic shuttling controls chondrocyte differentiation and is dependent on the Ca2+/calmodulin signaling pathway. Specific Aims 1: To determine whether activation of the Ca2+/calmodulin signaling pathway prevents nuclear entry of HDAC4 and enhances the binding of HDAC4 to the cytoplasmic binding protein 14-3-3. This may impair HDAC4-mediated inhibition of chondrocyte differentiation in the nucleus. This aim consists of the four sub-aims. Specific Aim 1-1: We will determine whether HDAC4 shuttling from the nucleus to the cytoplasm is dependent on CaMKIV signaling during chondrocyte differentiation. Specific Aim 1-2: We will determine whether HDAC4 associates with 14-3-3 proteins in a phosphorylation-dependent manner in chondrocytes. Specific Aim 1-3: We will determine whether constitutively active CaMKIV overcomes HDAC4-induced inhibition of Runx2 and allows chondrocyte differentiation to proceed. Specific Aim 1-4: We will determine whether changing CaMKIV levels affects the growth plate proliferation and differentiation in organ culture. Hypothesis 2: P38 MAPK activity controls chondrocyte hypertrophy by increasing caspase-regulated degradation of HDAC4, which releases Runx2 from a repressive influence of HDAC4. This aim consists of the following five sub-aims. The experimental flowchart is described in the figure below. Specific Aims 2: To determine if caspases induce the degradation of HDAC4 is controlled by p38 MAPK by using constitutively active MKK6 to elevate p38 and dominant negative p38 MAPK to repress p38 in the presence or absence of caspase inhibitors, which in turn increases Runx2 activity. This aim consists of the five sub-aims. Specific Aim 2-1: We will determine whether increase or decrease of chondrocyte p38 activity by transfection of constitutively active MKK6 (CaMKK6) or dominant negative p38 MAPK (DN p38) will affect caspase activity. Specific Aim 2-2: We will determine whether degradation of HDAC4 at Asp-289 site is dependent on caspase 2 and 3 in chondrocytes in the presence and absence of caspase 2 and 3 inhibitors. Specific Aim 2-3: We will determine whether chondrocyte differentiation is regulated by p38 activity. Elevation of chondrocyte p38 activity by constitutively active MKK6 (CaMKK6) and repression of chondrocyte p38 activity by dominant negative p38 MAPK (DN p38) will be employed in the presence or absence of caspase 2 and 3 inhibitors. Specific Aim 2-4: Expression of Runx2 mRNA will be examined by in situ hybridization with p38 MAPK defective growth plates from dominant negative p38 transgenic mice (DN p38). This examination will show whether the expression of Runx2 is dependent on chondrocyte p38 MAPK signaling. Specific Aim 2-5: The activities of MAPK, including p38, ERK, and JNK will be determined by Western blot to detect whether other MAPK are involved in the process. Such experiments will also be performed with p38 MAPK defective chondrocytes from DN p38 mice to determine whether these processes are dependent on chondrocyte p38 MAPK signaling. Significance. Although many factors have been found to impact the function of the growth plate, its biology remains poorly understood. The importance of clearly delineating the physiological controls of the physis cannot be overemphasized. Such knowledge could allow the prevention or correction of a number of bone-growth related problems in adults and children. Leg length discrepancy and dwarfism are just a few clinical dilemmas that may be provided with more elegant solutions if the molecular triggers and stops to the growth plate can be elucidated. The understanding of cell differentiation in growth plate may also be important to the understanding of abnormal chondrocyte differentiation in osteoarthritis.